1. Field of the Invention
The present invention is related to a method for detecting compounds that inhibit protein aggregation, wherein the method utilizes an in vitro fluorescence-based assay. The method of the present invention identifies compounds that have potential against deleterious conditions wherein protein aggregation is important to the pathology or function of the condition. In one embodiment, the present invention is related to a method for detecting compounds that inhibit viral assembly, thus identifying potential antiviral compounds.
2. Description of the Background Art
A virus is a disease-causing agent that contains nucleic acid and can alternate between an intracellular state and an extracellular state; however, viruses require a host cell's machinery for protein synthesis and therefore can only multiply in an intracellular state. In either state, viruses are submicroscopic particles containing either deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) surrounded by protein and occasionally containing other components. Viruses can vary widely in size, shape, chemical composition, range of organisms attacked, kinds of cell damage induced, and range of genetic capabilities. Viruses are known to infect animals, plants, bacteria, and fungi.
The structures (size and shape) of viruses are exceedingly diverse. The internal structure of viruses, however, generally has the nucleic acid located in the center surrounded by a protein coat called the capsid. The individual proteins that make up the capsid are called protein subunits, or capsomeres.
Replication of viruses requires that the virus induce a living cell of a host organism to make more of the essential components needed by the virus particle for growth. These components must then be assembled in the proper order, and the new virus particles must escape from the cell and infect other cells. The various phases of this replication process can be summarized as: 1) attachment (adsorption) of a virus particle to a sensitive cell; 2) penetration into the cell by the virus or its nucleic acid; 3) replication of the viral nucleic acid; 4) production of capsid protein and other essential viral constituents; 5) assembly of nucleic acid and capsid protein into new virus particles (or packaging of the virus particle); and 6) release of mature virus particles from the cell.
Given the severity of many viral infections in all types of hosts, a need always exists for new methods of treating and/or preventing deleterious viral infections. Based on the life cycle described herein before, one should be able to attack the progression of viral infection in an animal, plant, bacteria or fungi by interfering with (or inhibiting) any of the stages of the viral life cycle essential to viral infection or replication. Examination of particular viruses could lead to the discovery of such methods, which in turn could lead to the elucidation of a method applicable to all or nearly all viruses.
Most antiviral agents available today are traditional enzyme inhibitors. These include nucleotide analogs that affect replication (e.g., acylcovir and AZT) and protease inhibitors that affect processing of viral polyproteins. There are also polio- and rhinovirus specific small molecules that function by stabilizing the capsid, inhibiting release of viral nucleic acid.
Since the protein coat (also referred to interchangeably herein as a “capsid”) of viruses is critical to virus propagation, compounds that interfere with normal assembly of the viral protein coat, either by inhibiting assembly or inappropriately enhancing assembly (including but not limited to, accelerating assembly kinetics, stabilizing capsids, and misdirecting assembly), would potentially function as antiviral compounds. However, prior to the present invention, an appropriate assay has not been available for identifying compounds that inhibit, enhance and/or misdirect viral coat assembly. It is to such method of detecting compounds that interfere with viral coat assembly, in a sensitive and reproducible in vitro format that is compatible with high throughput screening, that the present invention is directed.